Regulation of extrathymic T cell development and turnover by oncostatin M

Chronic exposure to oncostatin M (OM) has been shown to stimulate extrathymic T cell development. The present work shows that in OM transgenic mice, 1) massive extrathymic T cell development takes place exclusively the lymph nodes (LNs) and not in the bone marrow, liver, intestines, or spleen; and 2) LNs are the sole site where the size of the mature CD4+ and CD8+ T cell pool is increased (6- to 7-fold). Moreover, when injected into OM transgenic mice, both transgenic and nontransgenic CD4+ and CD8+ T cells preferentially migrated to the LNs rather than the spleen. Studies of athymic recipients of fetal liver grafts showed that lymphopoietic pathway modulated by OM was truly thymus independent, and that nontransgenic progenitors could generate extrathymic CD4+CD8+ cells as well as mature T cells under the paracrine influence of OM. The progeny of the thymic-independent differentiation pathway regulated by OM was polyclonal in terms of Vbeta usage, exhibited a phenotype associated with previous TCR ligation, and displayed a rapid turnover rate (5-bromo-2'-deoxyuridine pulse-chase assays). This work suggests that chronic exposure to OM 1) discloses a unique ability of LNs to sustain extrathymic T cell development, and 2) increases the number and/or function of LN niches able to support seeding of recirculating mature T cells. Regulation of the lymphopoietic pathway discovered in OM transgenic mice could be of therapeutic interest for individuals with thymic hypoplasia or deficient peripheral T cell niches.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

CCR10 is important for the development of skin-specific gammadeltaT cells by regulating their migration and location

Unlike conventional αβ T cells, which preferentially reside in secondary lymphoid organs for adaptive immune responses, various subsets of unconventional T cells, such as the γδ T cells with innate properties, preferentially reside in epithelial tissues as the first line of defense. However, mechanisms underlying their tissue-specific development are not well understood. We report in this paper that among different thymic T cell subsets fetal thymic precursors of the prototypic skin intraepithelial Vγ3(+) T lymphocytes (sIELs) were selected to display a unique pattern of homing molecules, including a high level of CCR10 expression that was important for their development into sIELs. In fetal CCR10-knockout mice, the Vγ3(+) sIEL precursors developed normally in the thymus but were defective in migrating into the skin. Although the earlier defect in skin-seeding by sIEL precursors was partially compensated for by their normal expansion in the skin of adult CCR10-knockout mice, the Vγ3(+) sIELs displayed abnormal morphology and increasingly accumulated in the dermal region of the skin. These findings provide definite evidence that CCR10 is important in sIEL development by regulating the migration of sIEL precursors and their maintenance in proper regions of the skin and support the notion that unique homing properties of different thymic T cell subsets play an important role in their peripheral location.     

Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4(+)CD25(+) regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4(+)CD25(+) regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4(+)CD25(+) thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.     

Lymphocyte lineages at mucosal effector sites: rat salivary glands

Development of T cell lineages and the role of the thymus as a source of immature T cells in parotid (PG) and submandibular salivary glands (SMG) were studied in Fischer 344 rats using the Thy-1/CD45RC/RT6 expression model. In addition, the phenotypes of salivary gland lymphocytes were compared with other conventional and extrathymic populations. PG mononuclear cells consisted of T cells (38%), B cells (29%), and NK cells (4%). SMG had 19% T cells, 7% B cells, 37% NK cells, and an unusual population of CD3(-)/RT6(+) cells. In comparison with lymph node (LN), both PG and SMG were enriched in immature (Thy-1(+)) and activated (Thy-1(-)/CD45RC(-)/RT6(-)) T cells. Unchanged percentages of Thy-1(+) T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source. In contrast, thymectomy eliminated LN recent thymic emigrants. SMG had T cells with characteristics of extrathymic populations, expressing TCRgammadelta(+) (28%), the CD8alphaalpha homodimer (11%), and NKR-P1A (66%). Many SMG T cells expressed integrin alpha(E)beta(7). PG T cells resembled those isolated from LN in respect to TCR and CD8 isoform usage, but were enriched in alpha(E)beta(7)(+) T cells and in NKT cells. Thus, salivary gland mononuclear cells are composed of a variety of subpopulations whose distributions differ between SMG and PG and are distinct from LN. These studies provide a basis for further investigation of regionalization in the mucosal immune network and are relevant to the design of vaccine regimens and intervention during pathological immune processes.     

Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4(+)CD25(+) T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4(+)CD25(+) regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA x DO11.10 mice had increased numbers of CD4(+)CD25(+) regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA x DO11.10 mice, T cells expressing endogenous TCR alpha beta chains were CD4(+)CD25(-) T cells, whereas T cells expressing autoreactive TCR were selected as CD4(+)CD25(+) T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA x DO11.10 mice. In contrast, in DO11.10 mice, CD4(+)CD25(+) T cells expressed endogenous TCR alpha beta chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4(+)CD25(+) T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4(+)CD25(+) T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4(+)CD25(-) T cells from autoreactive T cell repertoire.     

Peripheral T lymphocytes recirculating back into the thymus can mediate thymocyte positive selection

The thymus continuously produces T lymphocytes that contribute to the maintenance of the peripheral T cell pool. Since peripheral recirculating T cells represent a very minor population among total thymocytes in normal animals, the relationship between the thymus and secondary lymphoid organs is generally considered unidirectional. Recently, several reports have described the presence of recirculating T cells in the thymus, raising issues regarding their possible function. In this article, we show that the niche for recirculating T cells in the thymus, i.e., their absolute number, is the same in lymphopenic and normal mice. Using a novel combination of TCR-transgenic mice in which the ligand necessary for positive selection of host T cells is only expressed by transferred donor T cells, we show that mature T cells recirculating back to the thymus can mediate positive selection.     

Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8 alpha alpha intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8 alpha alpha T cells. However, when TCR(+) thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8alphaalpha iIELs, while TCR(-) T cell progenitors did not. The TCR V beta 1 repertoire of CD8 alpha alpha(+) TCR V beta 1(+) iIELs contained only part of the TCR V beta 1 repertoire of total iIELs, and it exhibited no new members compared with CD8(+) T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8 alpha alpha iIELs originate in the thymus, T cells acquire the expression of CD8 alpha alpha homodimers in the gut microenvironment.     

ZAP-70 restoration in mice by in vivo thymic electroporation

Viral and non-viral vectors have been developed for gene therapy, but their use is associated with unresolved problems of efficacy and safety. Efficient and safe methods of DNA delivery need to be found for medical application. Here we report a new monopolar system of non-viral electro-gene transfer into the thymus in vivo that consists of the local application of electrical pulses after the introduction of the DNA. We assessed the proof of concept of this approach by correcting ZAP-70 deficient severe combined immunodeficiency (SCID) in mice. The thymic electro-gene transfer of the pCMV-ZAP-70-IRES-EGFP vector in these mice resulted in rapid T cell differentiation in the thymus with mature lymphocytes detected by three weeks in secondary lymphoid organs. Moreover, this system resulted in the generation of long-term functional T lymphocytes. Peripheral reconstituted T cells displayed a diversified T cell receptor (TCR) repertoire, and were responsive to alloantigens in vivo. This process applied to the thymus could represent a simplified and effective alternative for gene therapy of T cell immunodeficiencies.     

Molecular and genetic parameters defining T-cell clonal selection

Clonal selection of T cells occurs in the thymus and is responsible for generating a useful and functional repertoire of T cells. Aberrations in clonal selection result in altered T-cell homeostasis in the secondary lymphoid organs ranging from an absence of T cells to an overabundance of autoreactive T cells. The advent of new technologies facilitating the manipulation of the mouse genome has helped refine our understanding of the molecular and genetic pathways involved in clonal selection and has also revealed a high degree of complexity. Herein, we attempt to review recent advances in thymic selection processes, achieved mostly through genetic manipulations.     

Generating intrathymic microenvironments to establish T-cell tolerance

Alphabeta T cells pass through a series of lymphoid tissue stromal microenvironments to acquire self tolerance and functional competence. In the thymus, positive selection of the developing T-cell receptor repertoire occurs in the cortex, whereas events in the medulla purge the system of self reactivity. T cells that survive are exported to secondary lymphoid organs where they direct first primary and then memory immune responses. This Review focuses on the microenvironments that nurture T-cell development rather than on T cells themselves. We summarize current knowledge on the formation of thymic epithelial-cell microenvironments, and highlight similarities between the environments that produce T cells and those that select and maintain them during immune responses.     

Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and -dependent T cells in the gut

Whereas most T cells arise in the thymus, a distinct lineage of extrathymically derived T cells is present in the gut mucosa. The developmental origin of extrathymic T cells is poorly understood. We show here that Notch-1, a transmembrane receptor involved in T cell fate specification of bipotential T/B precursors in the thymus, is absolutely required for the development of extrathymic (as well as thymus-derived) mature T cells in the intestinal epithelium. In the absence of Notch-1, CD117(+) T cell precursors are relatively more abundant in the gut than the thymus, whereas immature B cells accumulate in the thymus but not the gut. Collectively, these data demonstrate that Notch-1 is essential for both thymic and extrathymic T cell fate specification and further suggest that bipotential T/B precursors that do not receive a Notch-1 signal adopt a B cell fate in the thymus but become developmentally arrested in the gut.     

Local glucocorticoid production in lymphoid organs of mice and birds: Functions in lymphocyte development

Circulating glucocorticoids (GCs) are powerful regulators of immunity. Stress-induced GC secretion by the adrenal glands initially enhances and later suppresses the immune response. GC targets include lymphocytes of the adaptive immune system, which are well known for their sensitivity to GCs. Less appreciated, however, is that GCs are locally produced in lymphoid organs, such as the thymus, where GCs play a critical role in selection of the T cell antigen receptor (TCR) repertoire. Here, we review the roles of systemic and locally-produced GCs in T lymphocyte development, which has been studied primarily in laboratory mice. By antagonizing TCR signaling in developing T cells, thymus-derived GCs promote selection of T cells with stronger TCR signaling. This results in increased T cell-mediated immune responses to a range of antigens. We then compare local and systemic GC patterns in mice to those in several bird species. Taken together, these studies suggest that a combination of adrenal and lymphoid GC production might function to adaptively regulate lymphocyte development and selection, and thus antigen-specific immune reactivity, to optimize survival under different environmental conditions. Future studies should examine how lymphoid GC patterns vary across other vertebrates, how GCs function in B lymphocyte development in the bone marrow, spleen, and the avian bursa of Fabricius, and whether GCs adaptively program immunity in free-living animals.     

The dendritic cell-specific CC-chemokine DC-CK1 is expressed by germinal center dendritic cells and attracts CD38-negative mantle zone B lymphocytes

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA(+) T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis demonstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38(-)) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation.     

The thymus is a common target organ in infectious diseases

Infectious disease immunology has largely focused on the effector immune response, changes in the blood and peripheral lymphoid organs of infected individuals, and vaccine development. Studies of the thymus in infected individuals have been neglected, although this is progressively changing. The thymus is a primary lymphoid organ, able to generate mature T cells that eventually colonize secondary lymphoid organs, and is therefore essential for peripheral T cell renewal. Recent data show that normal thymocyte development and export can be altered as a result of an infectious disease. One common feature is the severe atrophy of the infected organ, mainly due to the apoptosis-related depletion of immature CD4+CD8+ thymocytes. Additionally, thymocyte proliferation is frequently diminished. The microenvironmental compartment of the thymus is also affected, particularly in acute infectious diseases, with a densification of the epithelial network and an increase in the deposition of extracellular matrix. In the murine model of Chagas disease, intrathymic chemokine production is also enhanced, and thymocytes from Trypanosoma cruzi-infected mice exhibit greater numbers of cell migration-related receptors for chemokines and extracellular matrix, as well as increased migratory responses to the corresponding ligands. This profile is correlated with the appearance of potentially autoreactive thymus-derived immature CD4+CD8+ T cells in peripheral organs of infected animals. A variety of infectious agents--including viruses, protozoa, and fungi--invade the thymus, raising the hypothesis of the generation of central immunological tolerance for at least some of the infectious agent-derived antigens. It seems clear that the thymus is targeted in a variety of infections, and that such targeting may have consequences on the behavior of peripheral T lymphocytes. In this context, thymus-centered immunotherapeutic approaches potentially represent a new tool for the treatment of severe infectious diseases.     

The distribution of rapidly and slowly renewed T, B, and "null" lymphocytes in mouse bone marrow, thymus, lymph nodes, and spleen.

Combined radioautography and immunofluorescence were employed to discern the proportions of rapidly renewed (RR) and slowly renewed (SR) T, B, and null cells in mouse lymph nodes (LN), spleen (Spl), thymus (Thy), and the lymphocyte-rich fraction of bone marrow (BML). Thy and BML were found to consist predominantly of cells with a rapid turnover rate (95.4 and 76.9%, respectively) in accord with their roles as primary lymphoid organs. In contrast, the secondary lymphoid organs were primarily composed of SR cells (LN, 73.4%; Spl, 67.8%) and contained a more even admixture of the six lymphocyte subsets. Most cells in Thy were RR T cells (93.3%), whereas the predominant lymphocyte subpopulation in both LN and Spl consisted of SR T cells (56 and 50%, respectively), with RR T cells being much less frequent (14.6% LN; 6.8% Spl). BML contained both RR and SR cells which stained with the T-cell reagent (7.8 and 7.1%). These percentages are probably overestimates, however, since this reagent stained some nonlymphoid cells in BML. RR B cells were most plentiful in BML (31.4%) and Spl (23.3%), less common in LN (11.2%), and rare in Thy (0.4%). SR B cells were equally numerous in Spl (16%), LN (15.4%), and BML (15.3%). RR null cells were the most prevalent cell type in BML (37.7%), but were infrequent in other tissues (1.6% Thy, 2.1% LN, 2.1% Spl). SR null cells were rare in all tissues (0% Thy, 2% LN, 2.1% Spl, 0.7% BML). These experiments represent the first comprehensive investigation of the composition of the lymphomyeloid organs in terms of RR and SR T, B, and null cells. They conclusively demonstrate RR and SR lymphocytes in all three functional categories (T, B, and null) and show characteristic tissue distributions of the six lymphocyte subsets.     

Receptor revision in peripheral T cells creates a diverse V beta repertoire

In Vbeta5 transgenic mice, the age-dependent accumulation of Vbeta5(-)CD4(+) T cells expressing endogenous Vss elements represents an exception to the rule of strict allelic exclusion at the TCRbeta locus. The appearance of these cells is limited to the lymphoid periphery and is driven by a peripherally expressed tolerogen. Expression of the lymphoid-specific components of the recombinase machinery and the presence of recombination intermediates strongly suggest that TCR revision rescues tolerogen-reactive peripheral T cells from deletion. Here, we report that the appearance of Vbeta5(-)CD4(+) T cells is CD28-dependent. In addition, we find that the TCR repertoire of this unusual population of T cells in individual Vbeta5 transgenic mice is surprisingly diverse, both at the level of surface protein and at the nucleotide level within a given family of V(D)Jbeta rearrangements. This faithful recreation of the nontransgenic repertoire suggests that endogenous Vbeta-expressing populations do not arise from expansion of an initially rare subset. Furthermore, the undersized N regions in revised TCR genes distinguish these sequences from those generated in the adult thymus. The diversity of the revised TCRs, the minimal mouse-to-mouse variation in the expressed endogenous Vbeta repertoire, the atypical length of junctional sequences, and the CD28 dependence of the accumulation of Vbeta5(-)CD4(+) T cells all point to their extrathymic origin. Thus, tolerogen-driven receptor revision in peripheral T cells can expand the TCR repertoire extrathymically, thereby contributing to the flexibility of the immune repertoire.